The KIT receptor has critical roles in several cell systems including hematopoiesis, the pigmentary system, gametogenesis, and in pacemaker cells of the gastrointestinal tract. Normal Kit receptor mediated functions include cell proliferation, cell survival, cell adhesion, cell migration, secretory responses and differentiation. In human neoplasia oncogenic activation of Kit has roles in gastrointestinal stromal tumors, mastocytosis/mast cell leukemia, acute myelogenous leukemia, a minor subset of melanomas and a subset of germ cell tumors. Kit receptor functions are mediated by kinase activation, receptor autophosphorylation and association with various signaling molecules and signaling cascades. How do receptor tyrosine kinases such as Kit mediate distinct cellular responses in different cell types during embryonic development and in the postnatal animal;and what are the requirements for oncogenic transformation in different cell types to produce cancer. We have produced mice containing knock-in point mutations, loss of function and gain of function mutations in the Kit receptor gene in mice which block distinct signaling cascades or which provide for oncogenic activation of Kit in distinct cell types and driving oncogenesis. Most gastrointestinal stromal tumors express the KIT receptor tyrosine kinase, and oncogenic KIT signaling drives GIST tumorigenesis. The principal genetic events responsible for the pathogenesis of GIST are thought to be gain-of-function mutations in the KIT gene or occasionally in the PDGFR alpha gene. Patients with familial GIST syndrome carry a germline KIT gain-of-function mutation. The observation of inherited KIT gain of function mutations in familial GIST provided us with a rationale for developing mouse models for this disease. By using a knock-in strategy, the Kit-V558 deletion mutation found in a familial GIST case was introduced into the mouse genome. Remarkably, heterozygous mutant KitV558 /+ mice provide a faithful mouse model for human familial GIST, and demonstrated that constitutive KIT signaling is necessary and sufficient for induction of GIST and hyperplasia of interstitial cells of Cajal (Sommer et al., 2003). These GIST mice provide an excellent tool to study the role of the KIT receptor GIST oncogenesis in vivo and for studies of targeted pharmacological intervention. The overall objective of this proposal is twofold, 1) to continue our investigations into the mechanism of Kit driven GIST oncogenesis and tumor progression, and 2) to investigate new treatment strategies for imatinib sensitive and imatinib resistant GIST. PUBLIC HEALTH RELEVANCE: The Kit receptor has roles in pigment formation, hematopoiesis, germ cell development and pacemaker cells of the gastrointestinal tract in interstitial cells of Cajal. Based on phenotypes of Kit mutant mice, the cellular responses which Kit may mediate appear to be quite diverse and include cell proliferation, cell survival (suppression of apoptosis/cell death), cell adhesion, migration, secretory responses and differentiation. Furthermore, Kit has roles in human cancer. The cancers which are associated with oncogenic activation of Kit include most importantly GIST, but oncogenic activation of Kit is also observed in mastocytosis, seminomas, a small subset of AMLs and a small subset of melanomas. How does the Kit receptor mediate these diverse outcomes? The Kit receptor is known to activate several distinct signaling cascades including phosphatidyl inositol-3 kinase (PI 3-kinase) and Src family kinases (SFK). Both signaling cascades have critical roles in receptor tyrosine kinase signaling and oncogenic transformation. Previous work from our laboratory brought to light that PI 3- kinase signaling is critical in male germ cell development, but had no other discernible roles in other cell types, whereas SFK signaling has roles in hematopoietic cell lineages, but not in germ cell development. These results highlight the critical importance of the cellular environment or cellular context in which the Kit receptor functions and that animal models are critical for elucidating the role and mechanisms of receptor tyrosine kinase signaling in different cell types. In most cancers oncogenic Kit receptor mutations occur in somatic cells. However, in rare occasions oncogenic Kit mutations are acquired in germ cells and are transmitted in the germ line. We have engineered a mutation found in a familial GIST case into the mouse genome by using homologous recombination approaches and a mouse strain which carries this mutation in the germline has been obtained. These mice recapitulate human familial GIST quite faithfully. These findings demonstrated that the Kit mutation is the initiating event in the development of familial and presumably non-familial GIST. Second they highlight the unique specificity of the mutant Kit receptor to produce GIST and not other cancers and this implies that the cellular machinery in GIST cells and their microenvironment is quite unique in supporting tumor formation and tumor maintenance. These GIST mice provide a unique opportunity to investigate the requirement for oncogenic Kit receptor signaling in GIST tumorigenesis and they provide an opportunity to evaluate second generation drugs which might be useful in the treatment of imatinib resistant GIST. As part of this proposal first we are investigating the temporal requirement for oncogenic Kit signaling in GIST tumorigenesis and GIST tumor progression as well as the development of oncogene addiction. Second we will investigate the mechanism of oncogenic Kit receptor activation. Today patients with GIST are successfully treated with the kinase inhibitor imatinib. However most patients eventually become resistant to drug treatment as a result of the acquisition of a secondary Kit mutation. Encouraged by our success to recapitulate familial GIST in mice we have produced mouse models for imatinib resistant GIST. We now will use these mice to evaluate new therapeutic strategies.